Biotechnology in the 21st century presents the possibility of new solutions to food, environment and health problems, with the ultimate object of promoting human prosperity. In recent years, the technology of using stem cells has been considered as a new way to treat incurable diseases. Formerly, organ transplantation, gene therapy, etc., were proposed for the treatment of incurable human diseases, but their use has not been achieved efficiently due to immunorejection, a small supply of organs, and insufficient knowledge of genes.
For this reason, with increasing interest in stem cell research, it has been recognized that totipotent stem cells having the ability to form all organs by proliferation and differentiation can not only treat most diseases but also fundamentally heal organ injuries. Also, many scientists have suggested the applicability of stem cells for the regeneration of all the organs and the treatment of incurable diseases, including Parkinson's disease, various cancers, diabetes and spinal damage.
Stem cells refer to cells having not only self-replicating ability but also an ability to differentiate into at least two types of cells, and can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells (MSCs).
Totipotent stem cells are cells having totipotent properties capable of developing into one perfect individual, and these properties are possessed by cells up to the 8-cell stage after the fertilization of an oocyte and a sperm. When these cells are isolated and transplanted into the uterus, they can develop into one perfect individual. Pluripotent stem cells, which are cells capable of developing into various cells and tissues derived from the ectodermal, mesodermal and endodermal layers, are derived from an inner cell mass located inside of blastocysts generated 4-5 days after fertilization. These cells are also called embryonic stem cells and can differentiate into various other tissue cells but cannot form new living organisms.
Multipotent stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature, 418: 41, 2002), and then also found in other various adult tissues (C. M. Verfaillie, Trends Cell Biol., 12: 502, 2002). In other words, although the bone marrow is the most widely known source of stem cells, the multipotent stem cells were also found in the skin, blood vessels, muscles and brains (J. G. Tomas et al., Nat. Cell Biol., 3: 778, 2001; M. Sampaolesi et al., Science, 301: 487, 2003; Y. Jiang et al., Exp. Hematol., 30: 896, 2002). However, stem cells in adult tissues, such as the bone marrow, are very rarely present, and such cells are difficult to culture without inducing differentiation, and thus difficult to culture in the absence of specifically screened media.
The reason why it is important to establish cell lines of such multipotent stem cells is because of the objectives of the research on the proliferation, lyophilization and characterization of stem cell lines, drug tests, and the autologous, allogeneic and xenogeneic transplantation of stem cell lines.
The equine is a mammal belonging to the Equidae (Genus Equus), and is one of the rare types of animals that have been domesticated by humans, along with dogs or cats. Equine industries, including the horse racing industry, are growing worldwide. In the horse racing industry, racehorses with a good pedigree are highly valuable. When such racehorses with a good pedigree are injured, the healing of the racehorses is problematic. Thus, the development of cell therapy for equine animals has received attention. Considering the size of horses, it is a major technical issue to reliably supply a large number of cells.
Therefore, isolation and characterization of stem cells derived from various equine tissues have become important issues in the stem cell field. Conventionally, there have been studies on stem cells from human and mouse tissues in various fields. However, studies on cell therapeutic agents for the treatment of bone, cartilage, tendon or muscle of equine animals, in particular racehorses, have not yet been sufficient, even though the need for the cell therapeutic agents is great.
There was a report on the isolation of stem cells from equine adipose tissues (Armando de Mattos Carvalho et al., Veterinary Immunology and Immunopathology, 132: 303, 2009). However, equine animals do not have much adipose tissue, unlike other mammals, and thus it is very difficult to obtain adipose tissue from equine animals. In addition, a method of obtaining adipose from individuals is invasive and causes pain. In the case of stem cells from equine bone marrow, the isolation of bone marrow is also performed using an invasive method, like that used for obtaining adipose. Also, due to the nature of racehorses, there is a distinct limitation in the isolation of stem cells from adipose tissue or bone marrow.
In the current state of technology, in order to use adult stem cells as cell therapeutic agents, it is required to standardize the culture conditions under which an undifferentiated state can be maintained. In addition, because adult stem cells isolated from tissues are present as a mixture of various kinds of cells, it is required to develop technology capable of culturing homogeneous adult stem cells on a mass scale. In particular, methods for isolating adult stem cells from tissues or blood generally include, for example, cell sorting utilizing antibodies for a number of surface antigens. However, this method has a shortcoming in that the surface antigens of adult stem cells should be known. In addition, a common surface antigen (hereinafter referred to as “marker”) for adult stem cells is not yet known. Also, various markers for adult stem cells have not been developed, and known markers for adult stem cells are expressed at different levels depending on the differentiation state of adult stem cells. Particularly, a system of sorting cells according to the expression level of the markers is expensive. Due to such shortcomings, the use of the cell sorting method has been greatly limited.
The placenta plays an important role in the development and survival of a fetus by supplying nutrients and oxygen thereto. Generally, the placenta is disposed of as medical waste after delivery. However, recent studies indicate that human amniotic tissue is a source rich in stem cells, and many studies on stem cells derived therefrom have been conducted. In clinical applications, amniotic tissue has effects on wound healing and retinal reconstitution. The amnion may possibly contain stem cells in a mixture with other monocytes and other stem cells. Under culture conditions for such mixed cells, the distribution of nutrients cannot be uniform, thereby causing heterogeneity in differentiation of cells. Conclusively, the problem that the cells cannot be produced as a homogeneous cell population serves as a fatal disadvantage, as when they are used as the therapeutic agent the actual effect may be different from the intended effect. Therefore, there is an urgent need for the development of effective culture technology that makes it possible to obtain homogeneous adult stem cells in a large quantity.